MdCSN5–MdIAMT module promotes anthocyanin accumulation by regulating IAA homeostasis in apple
Jiahu Zhang, Chen Wang, Haibo Wang, Ping He, Yuansheng Chang, Sen Wang, Wenyan Zheng, Nan Wang, Yongxu Wang, Qi Zou, Linguang Li, Xuesen Chen, Xiaowen He
TL;DR
This study identifies a gene module in apples that regulates anthocyanin accumulation by controlling auxin levels, offering new insights into plant hormone signaling.
Contribution
The discovery of the MdCSN5-MdIAMT module as a novel regulator of auxin homeostasis and anthocyanin biosynthesis in apples.
Findings
MdIAMT overexpression increases anthocyanin accumulation by regulating IAA homeostasis.
MdCSN5 interacts with MdIAMT to influence the auxin signaling pathway.
The MdCSN5-MdIAMT module promotes anthocyanin accumulation in apple fruit.
Abstract
The apple anthocyanin content is an important trait in apple breeding. Auxin, as an important plant hormone, plays significant roles in regulating the biosynthesis of anthocyanins. However, the molecular mechanism of how plants regulate auxin content and activity to affect anthocyanin accumulation remains unclear. In this study, through fruit anthocyanin content analysis and transcriptome sequencing of the hybrids derived from ‘Golden Delicious’ and ‘Fuji Nagafu No. 2’ crosses, a key gene for regulating apple anthocyanin accumulation, indole-3-acetic acid (IAA) methyltransferase (MdIAMT), was identified. Functional analyses showed that the apple calli and peel overexpressing MdIAMT accumulated more anthocyanin than that in Vec by regulating IAA homeostasis. Yeast two-hybrid assays, luciferase complementation imaging assays and co-immunoprecipitation assays revealed that MdCSN5, an…
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Figure 7- —Earmarked Fund for China Agriculture Research System10.13039/501100010038
- —Shandong Provincial Postdoctoral Science Foundation10.13039/501100020196
- —Research and Innovation Program Youth Project of Shandong Institute of Pomology
- —Key Research and Development Program of Shandong Province10.13039/100014103
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TopicsPlant Gene Expression Analysis · Plant Molecular Biology Research · Horticultural and Viticultural Research
Introduction
In apple, as water-soluble flavonoid compounds, anthocyanins are not only the primary pigments responsible for the red coloration of apples, which promotes the quality of fruit, but also contributes to apple resistance to various stresses [1, 2]. Recent research has shown that the interaction between MdWRKY17 and MdWRKY50 can promote anthocyanin biosynthesis, increasing the drought tolerance of apple [3]. Low temperature increases the levels of MdMYB2, which binds the MdSIZ1 promoter and increases its expression, thereby promoting anthocyanin biosynthesis and thus increasing cold tolerance [4]. Therefore, enhancing the anthocyanin content in apple fruit is a crucial objective in apple breeding, and elucidating the mechanism of anthocyanin accumulation remains an important focus in apple research.
Anthocyanins are biosynthesized through the phenylalanine pathway, which involves the key enzymes phenylalanine ammonia lyase (PAL), chalcone isomerase (CHI), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), dihydroflavonol 4-reductase (DFR), and UDP-glucose: flavonoid 3-glucosyltransferase (UF3GT) [5]. Anthocyanin biosynthesis is regulated by multiple environmental factors, particularly light, temperature, moisture, nutrient availability, mechanical damage, and other factors [6]. Among them, light is the predominant environmental regulator of anthocyanin biosynthesis in plants, and anthocyanin biosynthesis is nearly completely inhibited in the absence of light [7]. In apple, light stimulates transcriptional activation of many structural genes and regulatory transcription factors in the anthocyanin biosynthesis pathway [8]. Research on the mechanism by which light promotes the synthesis of anthocyanins has received widespread attention. The COP9 signalosome (CSN) plays a pivotal role in light signaling by regulating various E3 ubiquitin ligases [9]. In the Arabidopsis thaliana csn5a mutant, anthocyanin levels are notably increased via regulating the differential expression of genes encoding proteins that compose TTG1/bHLH/MYB complexes [10, 11]. Conversely, heterologous overexpression of Glycine max CSN5A and CSN5B led to anthocyanin accumulation under phosphorus-deficient conditions [12]. The previous studies demonstrated that the regulatory effect of CSN5 on anthocyanin biosynthesis varies across different species. However, a homolog of CSN5 in apples has not yet been identified, and current understanding of how CSN5 participates in anthocyanin biosynthetic regulation remains unclear.
Methylation is a ubiquitous process that occurs in almost all organisms and plays an important regulatory role in numerous cellular processes [13]. The SABATH methyltransferase family is an important class of methyltransferases, which primary substrate is small molecules, especially plant hormones [14]. The first SABATH family member identified was the salicylate methyltransferase (SAMT) in Clarkia breweri, which can convert salicylic acid (SA) to methylated SA (MeSA) [15]. Methylated indole-3-acetic acid (MeIAA) is an inactive form of indole-3-acetic acid (IAA), and the in vivo activity of IAA depends on the generation of free IAA from MeIAA upon hydrolysis by plant esterizes [16]. IAA methyltransferase (IAMT) can methylate IAA to produce MeIAA, which was first identified in A. thaliana and has been demonstrated to serve as pivotal coordinators of growth regulation and auxin homeostasis [17]. In Picea crassifolia, the expression of PgIAMT1 dramatically changed during in vitro somatic embryo maturation, suggesting its potential involvement in embryogenesis via IAA homeostasis regulation [18].
Auxin is a key plant hormone that controls plant growth, development, and response to environmental changes [19]. The effect of auxin on anthocyanin biosynthesis depends on its concentration, the plant species, and the type of auxin [20]. Several studies have indicated that auxin inhibits anthocyanin accumulation in apples. Auxin treatment can induce the degradation of MdIAA121 to release MdARF13, which can directly bind to the promoter of MdDFR to inhibit anthocyanin biosynthesis [21]. Although there have been relevant reports on the regulation of anthocyanin biosynthesis by IAA in apples, the mechanism by which endogenous IAA homeostasis regulates anthocyanin biosynthesis in apples is still unclear.
In this study, MdIAMT, a methyltransferase gene, which influences apple anthocyanin biosynthesis by regulating IAA homeostasis, was identified. Furthermore, through a yeast two-hybrid (Y2H) screening assay, MdCSN5 was identified as a protein that interacts with MdIAMT. Functional analyses revealed that MdCSN5 regulates anthocyanin accumulation by modulating MdIAMT-mediated IAA homeostasis. Our findings revealed a novel mechanism of anthocyanin accumulation and holds great significance for apple breeding.
Results
Transcriptome profiling and validation of key genes governing anthocyanin accumulation in apple
Anthocyanins serve as the primary pigments responsible for red coloration in apple fruit [22]. The fruit peel colors of the F1 hybrid derived from ‘Golden Delicious’ and ‘Fuji Nagafu No. 2’ were highly different from the parental species. From the F1 hybrid population, the fruits of five red fruit peel (1–28, 1–70, 1–84, 3–76, 3–78) and five yellow fruit peel progenies (1–148, 1–165, 1–179, 3–136, 3–142) were collected at 10, 25, 55, 85, 115, and 145 days after full bloom (DAFB) (Fig. 1a and Fig. S1). The anthocyanin content did not obviously differ among the ‘Golden Delicious’, ‘Fuji Nagafu No. 2’, and F1 hybrid fruits, and was approximately 15–50 μg/g FW (fresh weight) from 10 to 115 DAFB. From 115 to 145 DAFB, the anthocyanin contents of the fruits with red peels significantly increased. At 145 DAFB, the anthocyanin contents of the fruits with red peels (1–28, 1–70, 1–84, 3–76, 3–78) increased from approximately 69.26 to approximately 194.90 μg/g FW (the hybrid 1–28 had the lowest content, the hybrid 1–70 had the highest content), while the anthocyanin contents of the fruits with yellow peels were all <25 μg/g FW (Fig. 1b).
Anthocyanin content determination and WGCNA analysis in selected F1 hybrids derived from ‘Golden Delicious’ and ‘Fuji Nagafu No. 2’ crosses. Representative phenotype (a), and anthocyanin content (b) of ‘Golden Delicious’, ‘Fuji Nagafu No. 2’, and F1 hybrids fruits on 10, 25, 55, 85, 115, and 145 DAFB. Error bars indicate the mean ± standard error of at least three independent experiments (n = 6). Scale bar = 2 cm. (c) Module–specimen associations. Each column represents a specimen and each row represents a module eigengene. The value in each cell at the row–column intersection represents the correlation coefficient between the module and the specimen, and the value in parentheses represents the P-value. (d) The heatmap and bar plot show the eigengene expression profiles of the greenyellow module.
To elucidate the molecular basis underlying apple anthocyanin content is regulated, transcriptome sequencing was performed on those 10 F1 hybrids. Following adapter trimming and quality filtering of raw reads, a total of 192.34 Gb of clean reads were generated. To conduct comprehensive analysis of 30 samples, weighted gene co-expression network analysis (WGCNA) was performed via TBtools [23]. All 46 558 genes in the 30 samples were analyzed, the expression levels of these genes were processed, and all the outliers were removed (Table S1). We calculated intergenic correlation coefficients and subsequently defined coexpression modules using clustering algorithms (Fig. S2). The expression levels of 46 558 genes were processed into 12 distinct gene coexpression modules (Fig. 1c, Table S2). Eigengene trait correlation analysis revealed that the genes in the greenyellow module were strongly correlated with the differences in peel color (Fig. 1c). The expression profiles of greenyellow module genes were visualized using a heatmap, while eigengene expression dynamics across sample groups (representing module expression pattern) were illustrated in a bar plot (Fig. 1d). The genes in the greenyellow module were expressed differently in the apples with red peels than in the apples with yellow peels, indicating that the genes identified in this module might play a vital role in regulating anthocyanin accumulation in apples.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to annotate the eigengenes in the greenyellow module. GO enrichment analysis revealed enriched biological process terms, including organophosphate metabolic process (GO:0019637), response to auxin (GO:0009733), and nucleotide metabolic process (GO:0009117) in the greenyellow modulate genes (Fig. 2a, Table S3). KEGG pathway enrichment analysis identified significant involvement of the candidate genes in flavonoid biosynthesis (mdm00941) and pentose phosphate pathway (mdm00030) (Fig. 2b, Table S4). These results suggested that these genes in the greenyellow module were significantly associated with anthocyanin biosynthesis.
Enrichment analyses of DEGs in F1 hybrids. GO (a) and KEGG (b) enrichment analysis of DEGs in greenyellow module. (c) Venn diagram indicating the overlapping DEGs identified in F1 hybrids. (d) KEGG enrichment analysis of 20 DEGs screened by Venn analysis.
To screen the key genes associated with anthocyanin accumulation in apple fruits, each red apple fruit was compared with every yellow apple fruit, and the numbers of differentially expressed genes (DEGs) are shown in Table S5–9. The DEGs from the term containing same red apple strain were subjected to further analyses. The results revealed 1471 DEGs associated with the RedA (1–28) hybrid, 983 DEGs associated with the RedB (1–70) hybrid, 922 DEGs associated with the RedC (1–84) hybrid, 637 DEGs associated with the RedD (3–76) hybrid, and 1228 DEGs associated with the RedE (3–78) hybrid (Fig. 2c). Through further analysis, 20 genes were found in all five hybrids (Fig. 2c, Table S10). A KEGG enrichment analysis of these 20 genes revealed that flavonoid biosynthesis (mdm00941) was the most enriched term (Fig. 2d, Table S11). The functions of these 20 genes were predicted and are shown in Table S10. Among them, MYB1 (MD09G1278600), F3H (MD06G1201700), ANS (MD03G1001100), and UF3GT (MD07G1306900) were considered as key genes involved in anthocyanin biosynthesis [24–26]. Collectively, we speculated that the identified 20 genes likely function as key regulators of anthocyanin biosynthesis.
MdIAMT positively regulates anthocyanin accumulation
By predicting the functions of these 20 identified genes, MD09G1254000 was screened for further analysis. Multiple sequence alignment revealed that MD09G1254000 had conserved SAM/SAH-binding motifs and most conserved IAA-binding motifs, such as Lysine^18^, Glutamine^33^, Tryptophan^158^, and Leucine^222^ (Fig. S3a). Phylogenetic analysis revealed that the identified SABATH family members with the same function clustered together and that MD09G1254000 was clustered close to other IAMTs; therefore, MD09G1254000 was named MdIAMT (Fig. S3b).
To verify that MdIAMT is the key gene regulating anthocyanin synthesis in the F1 hybrids, the expression levels of MdIAMT in the fruit peels of ‘Golden Delicious’, ‘Fuji Nagafu No. 2’ and the 10 F1 hybrids were detected at 145 DAFB by quantitative real-time polymerase chain reaction (RT-qPCR). The data demonstrated that transcriptional levels of MdIAMT were strongly correlated with the anthocyanin contents of the ‘Golden Delicious’, ‘Fuji Nagafu No. 2’, and F1 hybrid fruit peels at 145 DAFB (Fig. S4a, Table S12).
To verify whether MdIAMT could promote the anthocyanin accumulation in apples, MdIAMT-OE transgenic apple calli were generated and confirmed via western blotting and RT-qPCR (Fig. S5a and b). When cultured in the absence of light, the MdIAMT-OE transgenic apple calli were not different from Vec apple calli. After light treatment, the MdIAMT-OE transgenic apple calli had a deeper red color than the Vec apple calli (Fig. 3a). Moreover, the anthocyanin contents of the Vec and MdIAMT-OE transgenic apple calli were measured (Fig. 3b and c). The results revealed that anthocyanin accumulation was significantly induced by MdIAMT overexpression. Furthermore, the expression levels of anthocyanin synthesis-related genes (MdCHI, MdCHS, MdF3H, MdUF3GT) were measured via RT-qPCR, which revealed that the expression levels of anthocyanin synthesis-related genes increased in MdIAMT-OE transgenic apple calli (Fig. S6a).
MdIAMT positively regulated apple anthocyanin accumulation by influencing IAA homeostasis. Representative phenotype (a), anthocyanin content (b, c) of Vec and MdIAMT-OE transgenic apple calli exposed to light for 12 days. MeIAA (d), MeJA (e), and MeSA (f) contents of Vec and MdIAMT-OE transgenic apple calli exposed to light for 12 days using Plant MeIAA, Plant MeJA, and Plant MeSA ELISA Kits (Yuanji, Shanghai, China). (g) KEGG enrichment analysis of DEGs in MdIAMT-OE versus Vec comparisons. Representative phenotype (h), anthocyanin content (i, j) of Vec and MdIAMT-overexpression ‘Fuji’ apple fruits (MdIAMT-aOE) exposed to light for 6 days. Representative phenotype (k), anthocyanin content (l, m) of TRV::00 and MdIAMT-silenced ‘Fuji’ apple fruits (TRV::MdIAMT) exposed to light for 12 days. Error bars indicate the mean ± standard error of three independent experiments (n = 6). Different letters indicate significant differences (P < 0.05) based on Tukey’s HSD test.
To further verify whether MdIAMT is involved in phytohormone methylation, the contents of methyl phytohormones (MeIAA, MeSA, and methyl jasmonate (MeJA)) in Vec and *MdIAMT-*OE transgenic apple calli were measured, and the results revealed that the MeIAA content was significantly increased in MdIAMT-OE transgenic apple calli, whereas the methylation levels of other phytohormones (MeSA and MeJA) did not change significantly (Fig. 3d–f, Fig. S7a, Table S13). And the IAA contents of MdIAMT-OE transgenic apple calli were lower than Vec apple calli (Fig. S7b, Table S14). The SA and jasmonic acid (JA) contents of MdIAMT-OE transgenic apple calli showed no significant difference with Vec apple calli (Fig. S7c and d, Table S15–S16). To demonstrate the IAA methyltransferase activity of MdIAMT, the in vitro enzyme assays were performed. The MeIAA contents of the reaction mixture with MdIAMT protein were higher than with inactive MdIAMT protein, indicating that MdIAMT could catalyze IAA to produce MeIAA (Fig. S8, Table S17). Furthermore, to clarify the correlation between IAA content and anthocyanin content, the IAA content of F1 hybrid fruits was measured. At the 145 DAFB stage, the IAA content of F1 hybrid fruits was negative correlated with the content of anthocyanins (Fig. S9, Table S18).
To characterize the functional mechanism of MdIAMT in modulating anthocyanin accumulation in apple, a transcriptome sequencing analysis was performed between Vec and MdIAMT-OE transgenic apple calli. Comparing Vec with MdIAMT-OE calli, there were 2753 DEGs identified, with 1944 upregulated genes and 809 downregulated genes (Table S19). KEGG enrichment analysis revealed that the DEGs involved in phenylpropanoid biosynthesis (mdm00940), flavonoid biosynthesis (mdm00941), and plant hormone signal transduction (mdm04075), substantiating the function of MdIAMT in modulating anthocyanin accumulation in apple (Fig. 3g, Table S20).
A transient transgenic assay was also used to verify the role of MdIAMT in anthocyanin accumulation in apple fruit. The peels of apple fruit overexpressing MdIAMT (MdIAMT-aOE) were significantly red (Fig. 3h). Moreover, the anthocyanin contents of the apples overexpressing MdIAMT were significantly greater than those of the Vec apples (Fig. 3i–j). The gene expression levels of anthocyanin synthesis-related genes increased in apple fruits overexpressing MdIAMT, compared with that in Vec apples (Fig. S6b). A virus-induced gene silencing (VIGS) assay was used to silence MdIAMT expression in apple fruit. The results revealed that the peels of the apples with suppressed MdIAMT expression (TRV::MdIAMT) were yellow, whereas the Vec apples were red (Fig. 3k). The anthocyanin contents of the apples with silenced MdIAMT expression were significantly lower than those of the Vec apples (Fig. 3l and m). The expression levels of anthocyanin synthesis-related genes in the apples with silenced MdIAMT expression were lower than those in Vec apples (Fig. S6c). These results indicate that MdIAMT might promote anthocyanin accumulation by catalyzing IAA to produce MeIAA in apples.
MdIAMT interacts with MdCSN5
To explore the molecular mechanism of MdIAMT-mediated anthocyanin accumulation in apples, a Y2H screening assay was performed in which MdIAMT was used as the bait protein against a Malus domestica cDNA library to identify MdIAMT-interacting proteins. After removing duplicate genes, 11 genes were obtained, and MD08G1130400 was selected after functional analysis (Table S21). MD08G1130400 was isolated from the M. domestica cDNA library via PCR. Multiple-sequence alignment revealed that MD08G1130400 has a conserved MPN (Mpr1-Pad1-N-terminal) domain, similar to AtCSN5, the COP9 complex subunit, in other species, designated MdCSN5 (Fig. S10).
To confirm the interaction between MdIAMT and MdCSN5, a Y2H assay was performed. The yeast cells used as positive controls and transformed with the MdCSN5-BD and MdIAMT-AD plasmids could grow normally on SD-Leu/-Trp (DDO) or SD-Leu/-Trp/-His/-Ade (QDO) solid medium and become blue in color after x-α-gal was added to the QDO solid medium, whereas the yeast cells used as negative controls could not grow normally on the QDO solid medium (Fig. 4a). Additionally, protein–protein interactions between MdIAMT and MdCSN5 were validated using luciferase complementation imaging (LCI) assays. Fluorescence could be observed in the region with the MdCSN5-NLuc and MdIAMT-CLuc plasmids on the tobacco leaves but not in the negative control regions on the leaves (Fig. 4b). In the coimmunoprecipitation assay, the MdIAMT protein was immunoprecipitated by MdCSN5-MYC, which was consistent with the Y2H and LCI assay results (Fig. 4c). Taken together, these findings indicate that MdIAMT directly interacts with MdCSN5.
MdIAMT interacts with MdCSN5. (a) MdIAMT interacted with MdCSN5, based on the results of Y2H assays. The interaction of pGBKT7–53 with pGADT7-T was used as a positive control, and the interactions of pGBKT7-lam with pGADT7-T were used as negative controls. (b) Luciferase complementation imaging assay verifying the interaction between MdIAMT and MdCSN5 in N. benthamiana leaves. NLuc + CLuc: coexpression of a NLuc vector and a CLuc vector. NLuc + MdIAMT-CLuc: coexpression of an NLuc vector and MdIAMT-CLuc. MdCSN5-NLuc + CLuc: coexpression of MdCSN5-NLuc and a CLuc vector. MdCSN5-NLuc + MdIAMT-CLuc: coexpression of an MdCSN5-NLuc vector and an MdIAMT-CLuc vector. (c) Coimmunoprecipitation assay was performed to verify the interaction between MdCSN5 and MdIAMT.
MdCSN5 positively regulates anthocyanin accumulation
To investigate whether MdCSN5 plays a role in regulating anthocyanin accumulation in apples, the expression levels of MdCSN5 in the fruit peels of ‘Golden Delicious’, ‘Fuji Nagafu No. 2’,, and the 10 F1 hybrids were detected at 145 DAFB via RT-qPCR (Table S12). The results revealed that the expression levels of MdCSN5 were strongly correlated with the anthocyanin contents of ‘Golden Delicious’, ‘Fuji Nagafu No. 2’, and F1 hybrid fruit peels at 145 DAFB (Fig. S4b).
To verify that MdCSN5 can promote anthocyanin accumulation in apples, MdCSN5-overexpressing transgenic apple calli were constructed. Western blotting and RT-qPCR confirmed that MdCSN5 was specifically overexpressed in the transgenic apple calli (Fig. S11). Compared with Vec apple calli, MdCSN5-OE transgenic apple calli presented a deeper red color, a higher anthocyanin content, and the expression levels of anthocyanin synthesis-related genes were upregulated (Fig. 5a–c and Fig. S12a). To further verify whether MdCSN5 could promote the accumulation of anthocyanin in apples, a transient transformation assay was used to overexpress MdCSN5 in apple fruit (MdCSN5-aOE), and a VIGS assay was used to silence MdCSN5 in apple fruit (TRV::MdCSN5). A deep red color and high anthocyanin content was observed in the apples overexpressing MdCSN5 (Fig. 5d–f), whereas the apples with silenced MdCSN5 expression had a yellow color and anthocyanin contents less than those of the TRV::00 apples (Fig. 5g–i). The expression levels of anthocyanin biosynthetic genes increased in the apples overexpressing MdCSN5 but decreased in the apples silencing MdCSN5 (Fig. S12b and c). These results suggested that MdCSN5 promoted anthocyanin accumulation in apples.
MdCSN5 positively regulated apple anthocyanin accumulation. Representative phenotype (a), anthocyanin content (b and c) of Vec and MdCSN5-OE transgenic apple calli exposed to light for 12 days. Representative phenotype (d), anthocyanin content (e and f) of Vec and MdCSN5-overexpression ‘Fuji’ apple fruits (MdCSN5-aOE) exposed to light for 6 days. Representative phenotype (g), anthocyanin content (h and i) of TRV::00 and MdCSN5-silenced ‘Fuji’ apple fruits (TRV::MdCSN5) exposed to light for 12 days. Error bars indicate the mean ± standard error of three independent experiments (n = 6). Different letters indicate significant differences (P < 0.05) based on Tukey’s HSD test.
MdCSN5 regulates anthocyanin accumulation through MdIAMT-mediated homeostasis of IAA
To explore whether MdCSN5 works together with MdIAMT in regulating anthocyanin accumulation, a comparative transcriptome sequencing analysis was performed between Vec and MdCSN5-OE transgenic apple calli. A total of 3272 DEGs were identified comparing Vec with MdCSN5-OE calli, with 2662 genes exhibiting upregulation and 610 genes showing downregulation (Table S22). Subsequent integration of the transcriptome sequencing data from Vec and MdCSN5-OE transgenic apple calli with that of Vec and MdIAMT-OE transgenic apple calli facilitated a comprehensive analysis. Venn diagram analysis revealed 1109 genes upregulated and 194 genes downregulated in both the MdCSN5-OE versus Vec and MdIAMT-OE versus Vec comparisons (Fig. 6a). The KEGG enrichment analysis of these common DEGs underscored their involvement in key biological processes, specifically flavonoid biosynthesis (mdm00941) and plant hormone signal transduction pathways (mdm04075) (Fig. 6b, Table S23).
MdCSN5 regulates anthocyanin accumulation through MdIAMT-mediated homeostasis of IAA. (a) Venn diagram indicating the overlapping DEGs identified between MdIAMT-OE versus Vec and MdCSN5-OE versus Vec comparisons. (b) KEGG enrichment analysis of DEGs in both the MdIAMT-OE versus Vec and MdCSN5-OE versus Vec comparisons. (c) Content of MeIAA in Vec- and MdCSN5-overexpressing calli was measured using Plant MeIAA ELISA Kits (Yuanji, Shanghai, China). Representative phenotype (d), anthocyanin content (e, f) of Vec, MdCSN5-overexpression (MdCSN5-aOE), and MdCSN5-aOE with silenced MdIAMT (MdCSN5-aOE + MdIAMT-TRV) apple fruits exposed to light for 6 days. (g) Transcript levels of ARFs in Vec, MdIAMT-OE, and MdCSN5-OE transgenic apple calli exposed to light for 12 days. (h) Transcript levels of ARFs in Vec, MdIAMT-aOE, MdCSN5-aOE, and MdCSN5-aOE with silenced MdIAMT apple fruit exposed to light for 6 days. (i) Transcript levels of MdDFR in Vec and MdIAMT-OE transgenic apple calli exposed to light for 12 days. (j) Transcript levels of MdDFR in Vec and MdIAMT-aOE apple fruits exposed to light for 6 days. (k) Transcript levels of MdDFR in Vec and MdCSN5-OE transgenic apple calli exposed to light for 12 days. (l) Transcript levels of MdDFR in Vec and MdCSN5-aOE apple fruits exposed to light for 6 days. (m) Transcript levels of MdDFR in Vec, MdCSN5-aOE, and MdCSN5-aOE with silenced MdIAMT apples fruits exposed to light for 6 days. Error bars indicate the mean ± standard error of three independent experiments (n = 6). Different letters indicate significant differences (P < 0.05) based on Tukey’s HSD test.
The above results indicated that overexpressing MdIAMT increased MeIAA content in transgenic apple calli and affected IAA homeostasis. To explore whether MdCSN5 could affect contents of MeIAA in apple, the MeIAA contents of MdCSN5-OE transgenic apple calli were measured, and the result showed that the MeIAA contents of MdCSN5-OE transgenic apple calli were higher than Vec apple calli (Fig. 6c, Fig. S7a, Table S13). And the IAA contents of MdCSN5-OE transgenic apple calli were measured by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS), and the results showed that the IAA contents of MdCSN5-OE transgenic apple calli were lower than Vec apple calli (Fig. S7b, Table S14). Furthermore, the SA and JA contents of MdCSN5-OE transgenic apple calli showed no significant difference with Vec apple calli (Fig. S7c–d, Table S15–S16). To determine whether the increased anthocyanin accumulation in the apples overexpressing MdCSN5 depended on IAA homeostasis regulated by MdIAMT, a transient transformation assay was used to overexpress MdCSN5 and silence MdIAMT in apple fruit (MdCSN5-aOE + MdIAMT-TRV). The results revealed that the anthocyanin content of the apples overexpressing MdCSN5 with silenced MdIAMT was lower than that of the apples overexpressing only MdCSN5, indicating that MdCSN5 regulates anthocyanin accumulation through MdIAMT-mediated homeostasis of IAA (Fig. 6d–f).
Auxin response factors (ARFs) represent core transcriptional regulators in the auxin signaling [21]. To investigate the role of MdCSN5-MdIAMT module in promoting anthocyanin accumulation through the regulation of IAA homeostasis, the transcript levels of 18 ARFs were measured. The results indicated that the transcript levels of almost all ARFs (except MdARF9 and MdARF20) were significantly lower in MdIAMT-OE and MdCSN5-OE transgenic apple calli, compared to the Vec apple calli (Fig. 6g). And in apple peels that were transiently overexpressing MdIAMT and MdCSN5, the expression levels of most of these ARFs were also decreased (Fig. 6h). More notably, in apple peel overexpressing MdCSN5 with silenced MdIAMT, the transcript levels of the ARFs (except MdARF9 and MdARF16) returned to levels comparable to even higher than those in Vec (Fig. 6h). Furthermore, previous research had demonstrated that MdARF13 can negatively regulate the expression of anthocyanin biosynthesis gene MdDFR to inhibit anthocyanin biosynthesis [21]. Thus, the expression levels of MdDFR were measured. The transcripts of MdDFR were significantly increased in MdIAMT-OE and MdCSN5-OE transgenic calli compared to the Vec apple calli, as well as in apple peels that were transiently overexpressing MdIAMT and MdCSN5 (Fig. 6i–l). In apple peel overexpressing MdCSN5 with silenced MdIAMT, the transcript level of the MdDFR returned to the similarity level as Vec (Fig. 6m).
Discussion
The anthocyanin content of apple peels is a critical trait that significantly influences apple breeding strategies [27]. Consequently, investigating the mechanism of anthocyanin biosynthesis in apples has emerged as a prominent focus in apple molecular biology research [28]. Auxin is recognized as the main factors regulating anthocyanin biosynthesis; however, the molecular mechanism of how plants regulate auxin content and activity to affect anthocyanin accumulation remains poorly understood [20]. In this study, an important SABATH family gene, MdIAMT, which promotes anthocyanin accumulation by regulating IAA homeostasis in apples, was isolated. Further study revealed that MdCSN5, a component in light signal transduction, interacts with MdIAMT, promoting anthocyanin accumulation through MdIAMT-mediated regulation of IAA homeostasis in apples (Fig. 7). Our study revealed a novel mechanism by which auxin regulated anthocyanin accumulation via MdCSN5-MdIAMT module and provided new insights in the mechanism of plant hormone regulation of anthocyanin biosynthesis and in breeding high-anthocyanin-containing apple varieties.
Diagram depicting the mechanism of MdCSN5-MdIAMT module regulating anthocyanin accumulation in apple. MdCSN5 interacts with MdIAMT, and most of IAA are methylated to MeIAA, an inactive form of IAA. The expression levels of ARFs are decreased, and the inhibition of MdDFR expression from MdARF13 or other anthocyanin biosynthesis genes are weakened, leading to anthocyanin accumulation. ‘+’ means being enhanced. ‘–’ means being weakened. SAH, S-adenosine-L-homocysteine; SAM, S-adenosyl-L-methionine.
Transcriptome analysis is an important approach for elucidating the molecular mechanism of differential phenotypes [29]. WGCNA provides an effective computational framework for identifying trait-associated hub genes in plant [30]. To gain insight into the regulation mechanism of apple color, RNA-seq and WGCNA of three cultivars exhibiting differential peel coloration were performed, revealing that MdMYB28 could directly bind to MdMYB10 promoter to inhibit anthocyanin biosynthesis [31]. The transcriptomes of ‘Redchief Delicious’ apple fruits subjected to 25°C and 35°C treatments were used to explore the underlying mechanism by which high temperature inhibits anthocyanin accumulation, and after WGCNA, the results revealed that MdLBD37 is closely involved in the regulation of apple color [32]. In this study, WGCNA were used to analyze the key genes regulating anthocyanin accumulation in apple hybrids. The genes in the greenyellow module of WGCNA exhibited strong correlation with peel color variation (Fig. 1). Functional annotation through KEGG pathways demonstrated that the genes in this module are involved in flavonoid biosynthesis (mdm00941) and the pentose phosphate pathway (mdm00030) (Fig. 2). As anthocyanin is an important flavonoid [1], the genes in the greenyellow module were considered to be closely involved in anthocyanin biosynthesis. GO enrichment analysis revealed that the response to auxin (GO:0009733) was enriched in the greenyellow module genes (Fig. 2). In phytohormone-mediated anthocyanin biosynthesis, abscisic acid, ethylene, jasmonate, and cytokinins promote flavonoid biosynthesis, while auxin often play negative roles in regulating flavonoid biosynthesis [33]. The content of anthocyanin increased dramatically after auxin- and cytokinin-cotreated apples. However, excessive auxin concentration strongly hinders anthocyanin production even with the presence of cytokinin [34]. MYB10 was the key gene that promoted the biosynthesis of anthocyanins in red raspberry. And the expression of MYB10 was upregulated by MeJA and downregulated by IAA [35]. IAA has been widely shown to regulate anthocyanin biosynthesis and can inhibit anthocyanin biosynthesis through the Aux/IAA-ARF signaling pathway or IAA29-ARF5–1-ERF3 module in apples [21, 36]. Thus, the results of GO enrichment analysis further implied that auxin likely serves as a key regulator of anthocyanin accumulation in apple.
Subsequent analyses yielded a total of 20 candidate genes. KEGG enrichment analysis and functional prediction revealed that these genes were highly related to anthocyanin accumulation. MD09G1278600, identified as MdMYB1, has been widely confirmed as a crucial regulator of anthocyanin accumulation [37]. Various environmental factors, phytohormones and transcription factors regulate anthocyanin biosynthesis through MdMYB1 in apples [33]. Other important genes included MD06G1201700 (F3H), which encodes flavanone 3-hydroxylase, an enzyme that is responsible for the conversion of flavanones to dihydroflavonol [25]; MD03G1001100 (ANS), which encodes anthocyanidin synthase, an enzyme that catalyzes the conversion of leucoanthocyanidin to anthocyanidins [26]; and MD07G1306900 (UFGT), which encodes glycosyltransferase, an enzyme that catalyzes the conversion of anthocyanidins to anthocyanins [24]. F3H, ANS, and UFGT are indispensable for anthocyanin accumulation [28]. Overall, these 20 genes may be the key genes involved in anthocyanin accumulation.
The SABATH methyltransferase family is a critical group of methyltransferases that predominantly catalyze the conversion of carboxylic acids to their methyl ester forms [14]. The methylated compounds may acquire new biological properties and ecological roles distinct from their precursor molecules [38]. Previous studies have indicated that MeIAA is an inactive form of IAA, which more readily diffuses across membranes than IAA does [17]. The interconversion between IAA and MeIAA is highly important for the regulation of IAA homeostasis, which is critical for IAA function [16]. By predicting the functions of these 20 screened genes, MD09G1254000, an MdIAMT, was isolated. In F1 hybrids, MdIAMT expression levels and anthocyanin content were highly correlated, indicating that MdIAMT-mediated IAA homeostasis is crucial for anthocyanin accumulation in apples. Functional analysis revealed that MdIAMT promoted anthocyanin accumulation by regulating IAA homeostasis, proven by the results of transcriptome analysis of apple calli, providing a theoretical basis for apple fruit color regulation and a novel idea for apple breeding (Fig. 3).
Light serves as the predominant environmental regulator of anthocyanin biosynthesis in plants [7]. CSN5, a subunit of the CSN complex, can also independently affect anthocyanin accumulation [11]. The regulatory role of CSN5 in anthocyanin biosynthesis exhibits species-specific variation. In the A. thaliana csn5a mutant, anthocyanin biosynthesis is notably induced, which is related to increased MYB75 expression and suppressed GL2 expression [11]. In Solanum lycopersicum, SlCSN5–2 can increase the ubiquitination-mediated degradation of SlBBX20 to inhibit SlBBX20 binding to the anthocyanin biosynthesis gene SlDFR promoter and reduce anthocyanin biosynthesis [39]. Conversely, the Pi starvation-responsive GmCSN5A and GmCSN5B promoted anthocyanin accumulation in plants in response to P deficiency [12]. In this study, MdCSN5, which can interact with MdIAMT, was screened through a Y2H screening assay (Fig. 4). The results of functional assays indicated that, when cultured in the absence of light, the MdCSN5-OE transgenic apple calli were not different from the Vec apple calli, while MdCSN5-OE transgenic apple calli presented a deeper red color after light treatment than Vec apple calli. These results indicated MdCSN5 mediated the biosynthesis of anthocyanins under light. Notably, the anthocyanin contents of the apples with MdCSN5 overexpression and silenced MdIAMT expression were lower than those of apples overexpressing MdCSN5 only, indicating that MdCSN5 regulates anthocyanin accumulation through MdIAMT (Fig. 6). UPLC-MS/MS analysis revealed that the MeIAA contents in MdIAMT-OE and MdCSN5-OE transgenic apple calli exhibited significantly elevated levels compared to the control apple calli, while IAA contents were opposite, indicating that MdCSN5 regulates anthocyanin accumulation through MdIAMT-mediated IAA homeostasis regulation. ARFs are the key element of auxin signaling transduction pathway, auxin regulates plant growth and responses to various stresses by upregulating the expression of ARFs [21]. Recent research has shown that, under NAA treatment, MdSINA4, acts as an E3 ubiquitin ligase, targeted MdSINA11 and MdIAA29 for ubiquitin-dependent degradation, thereby releasing MdARF5–1, which inhibits MdERF3 expression to reduce anthocyanin biosynthesis [36]. To verify whether MdCSN5–MdIAMT module regulates anthocyanin accumulation through effecting auxin signaling transduction pathway, the transcript levels of 18 ARFs were measured. The results showed that overexpressing MdIAMT and MdCSN5 decreased the transcript levels of almost all detected ARFs (except MdARF9 and MdARF20). Furthermore, previous study indicated that MdARF13 inhibits the expression of anthocyanin biosynthesis gene MdDFR to negatively regulate anthocyanin accumulation in apple [21]. In this work, the expression of MdARF13 in overexpressing MdIAMT and MdCSN5 calli were downregulated while the expression level of MdDFR were upregulated (Fig. 6). These results indicated that MdCSN5–MdIAMT module-mediated IAA homeostasis regulates anthocyanin accumulation.
In conclusion, we have identified a novel anthocyanin accumulation mechanism in which MdCSN5 interacts with MdIAMT to promote anthocyanin accumulation via the regulation of IAA homeostasis was elucidated (Fig. 7). This pathway improved our understanding of the auxin-mediated regulation of anthocyanin accumulation, laying a foundation for breeding excellent apple varieties and optimizing green cultivation production technology.
Materials and methods
Plant materials and treatments
‘Golden Delicious’, ‘Fuji Nagafu No. 2’, and the F1 hybrid fruits obtained from cross-breeding the two varieties were picked at 10, 25, 55, 85, 115, and 145 DAFB from the Shandong Institute of Pomology base at Tai’an Tianping Lake (36°13′N, 117°01′E). ‘Fuji’ and ‘Red Delicious’ apples that were bagged were used for the transient transformation assay. The treatment conditions were a temperature of 16°C under a photosynthetic photon flux density (PPFD) of 100 μmol m^−2^ s^−1^. The cultured growth conditions of ‘Orin’ apple calli were 24 ± 0.5°C, 24 h darkness and a relative humidity of 60%–75%. The treatment conditions for the apple calli were a temperature of 16°C at 100 μmol m^−2^ s^−1^ PPFD. Nicotiana benthamiana were cultured under a 16-h light/8-h dark cycle at 23°C.
Measurement of total anthocyanin content and analysis of color differences
The anthocyanin contents of the fruits were measured using a Plant Anthocyanin Content Assay Kit (Grace Biotechnology, Suzhou, China) in accordance with the manufacturer’s guidelines and previous studies [40].
Measurement of MeIAA, MeJA, MeSA, IAA, JA, and SA contents
Fresh plant materials were homogenized with PBS in an ice bath. The extract was centrifuged at 1000 × g for 20 min, then we transferred the supernatant to a clean tube for further analysis. The MeIAA, MeJA, and MeSA contents were measured separately using Plant MeIAA, Plant MeJA, and Plant MeSA ELISA Kits (Yuanji, Shanghai, China) according to the manufacturer’s protocol.
The concentration of MeIAA, IAA, JA, and SA were also determined using a UPLC-MS/MS system. The samples from each replicate were ground in liquid nitrogen. The powder was extracted with 1.5 ml of extract solution (Methanol: H_2_O: Formic acid = 79.9: 20: 0.1, V/V/V), and the internal standard followed by vortex mixing for 1 min and ultrasonication for 30 min. For MeIAA and IAA, the D5-IAA (0.1 μg/ml) (OlchemIm s.r.o, Czech) was used as internal standard. For SA, salicylic acid (0.1 μg/ml) (Yuanye, China) was used as internal standard. For JA, jasmonic acid (0.1 μg/ml) (OlchemIm s.r.o, Czech) was used as internal standard. The extraction was kept mixing under 4°C for 16 h. After centrifugation at 10000 × g for 10 min, the supernatants were collected. A UPLC-MS/MS system (Hypersil Gold C18 column (3 μm, 2.1×100 mm)) was used to determine the concentrations. The column temperature was maintained at 35°C. Three biological replicates were analyzed for each experimental sample.
Transcriptome profiling and WGCNA
The red or yellow apple fruit peels from F1 hybrids were subjected to RNA-seq via the Illumina sequencing platform at Novogene Technologies (Beijing, China), as well as Vec, MdIAMT-OE, and MdCSN5-OE apple calli. Three biological replicates of each sample were analyzed for each experiment. Peels from the ripe fruits of F1 hybrids growing under normal growth conditions were sampled for RNA-Seq analyses. GDDH13 version 1.1 database was used as the reference genome, and the clean reads were queried via hierarchical indexing for spliced alignment of transcripts (HISAT) software at Novogene Technologies (Beijing, China). Genes that met the criteria of Padj < 0.05 and a fold change >2 were considered DEGs. The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) was used to determine the expression level of genes. WGCNA was performed via TBtools [23].
RNA extraction and quantitative real-time PCR analysis
Total RNA was isolated using Plant RNA Rapid Extraction Kit (Nobelab, Beijing, China). First-strand cDNA was synthesized via reverse transcription with R712 Rescript II RT SuperMix for qPCR (+gDNA Eraser) (Nobelab, Beijing, China). RT-qPCR was performed using the CFX96TM Real-Time System (Bio-Rad, USA) with Bestar™ qPCR MasterMix (SYBR Green) (DBI, Germany). The MdActin (XM_029089583.1) gene served as an internal control. Relative expression levels of the genes were calculated through 2^−ΔΔCt^ method. All primers used are listed in the Table S24.
Gene cloning and vector construction
The sequences of MdIAMT (MD09G1254000) and MdCSN5 (MD08G1130400) were amplified from the apples via PCR. The MdIAMT-OE recombinant vector was constructed by inserting Open Reading Frame (ORF) of MdIAMT into the pPZP211-3FLAG vector, and the MdCSN5-OE recombinant vector was constructed by inserting ORF of MdCSN5 into the pCAMBIA1305-3MYC vector for transgenic overexpression assays. The MdIAMT-TRV and MdCSN5-TRV recombinant vectors were constructed by inserting selected fragments of MdIAMT (411–803 bp) or MdCSN5 (451–936 bp) into the TRV2 vector for the VIGS assay. The MdIAMT-AD recombinant vector was constructed by inserting ORF of MdIAMT into the pGADT7 vector, and the MdCSN5-BD recombinant vector was constructed by inserting ORF of MdCSN5 into the pGBKT7 vector for the Y2H assay. The MdIAMT-CLuc recombinant vector was constructed by inserting ORF of MdIAMT into the pCAMBIA1300-CLuc vector, and the MdCSN5-NLuc recombinant vector was constructed by inserting ORF of MdCSN5 into the pCAMBIA1300-NLuc vector for LCI assay. ORF of MdIAMT was inserted into pGEX4T-1 to construct MdIAMT-GST recombinant vector for enzyme assays. All primers used are listed in the Table S24.
Transient overexpression and VIGS of apple fruits
The MdIAMT-OE and MdCSN5-OE recombinant vectors were transformed into Agrobacterium tumefaciens GV3101 for transient overexpression. The MdIAMT-TRV and MdCSN5-TRV recombinant vectors were transformed into A. tumefaciens GV3101 for VIGS. The ‘Fuji’ and ‘Red Delicious’ apple fruits were bagged and placed in the dark for 24 h after picking. Syringes (1 ml) were used to inject A. tumefaciens cells into apple fruits. The treated fruits were placed in the dark for 24 h and then transferred to an incubator at 16°C with 100 μmol m^−2^ s^−1^ PPFD.
Stable transformation of apple calli
The MdIAMT-OE and MdCSN5-OE recombinant vectors were transformed into A. tumefaciens LBA4404. Suitable apple calli were cultured with A. tumefaciens for 20 min and then incubated in Murashige and Skoog (MS) culture medium in the dark for 48 h. Medium containing antibiotics was used to select the transformed calli. Transgenic apple calli were confirmed by western blotting and RT-qPCR.
Enzyme assays
The MdIAMT-GST recombinant vectors were subsequently transformed into Escherichia coli strain Rosetta (DE3) (Weidi, Shanghai, China) for expression. The recombinant protein was purified through GST-tag Protein Purification Kit (Beyotime, Shanghai, China). The reaction volume was 200 μl, containing Tris–HCl buffer (50 mM, pH 8.0), 1 mM SAM, 200 μM IAA, and 25 μl purified protein as descripted by Liao et al. [41]. Enzymes were boiled at 100°C and used as controls. The reaction was incubated at 25°C for 4 h. The concentration of MeIAA was determined using a UPLC-MS/MS system.
Yeast two-hybrid assay
A Y2H screening assay of the M. domestica cDNA library was performed using MdIAMT as the bait protein to identify the target interact proteins. The experiment was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech, USA) according to the manufacturer’s protocol.
The MdIAMT-AD and MdCSN5-BD recombinant vectors were subsequently transformed into Y2H GOLD yeast strain. pGADT7-T and pGBKT7-53 vectors were used as positive controls, and pGADT7-T and pGBKT7-Lam vectors were used as negative controls. SD-Trp/-Leu (DDO) solid medium was used to culture the transformed yeast cells for 3–5 d, and then the transformed yeast cells were transferred to SD-Leu/-Trp/-His/-Ade (QDO) solid media and grown for 3–5 d. x-α-gal was used for the false positive test.
LCI assays
The MdIAMT-CLuc, MdCSN5-NLuc, pCAMBIA1300-CLuc (C-LUC) and pCAMBIA1300-NLuc (N-LUC) vectors were transformed into A. tumefaciens GV3101 for the LCI assay. Three combinations of A. tumefaciens cells were used as negative controls: C-LUC and N-LUC, MdIAMT-CLuc and N-LUC, C-LUC and MdCSN5-NLuc. Syringes (1 ml) were used to inject various vector combinations into Nicotiana benthamiana leaves as descripted by Wang et al. [42]. Fluorescence was observed via a PlantView living plant imaging system (Bltlux, Guangzhou, China).
Statistical analyses
The experiments in this work were independently replicated in triplicate. Data are presented as the mean ± standard error of the mean (SEM). Differences among samples were performed using one-way analysis of variance (ANOVA) with Tukey’s test via SPSS statistics software.
Accession numbers
The sequence data of genes detected by RT-qPCR in this study can be found through following accession numbers: MdIAMT (MD09G1254000), MdCSN5 (MD08G1130400), MdCHI (MD07G1186300), MdCHS (MD04G1003300), MdDFR (MD15G1024100), MdF3H (MD15G1246200), MdUF3GT (MD01G1234400), MdARF3 (MD05G1309400), MdARF5 (MD15G1014400), MdARF6 (MD05G1279200), MdARF7 (MD09G1145000), MdARF8 (MD04G1096900), MdARF9 (MD17G1272200), MdARF10 (MD06G1132100), MdARF13 (MD11G1132400), MdARF14 (MD14G1148500), MdARF15 (MD01G1108100), MdARF16 (MD07G1174000), MdARF17 (MD00G1103900), MdARF18 (MD15G1359400), MdARF19 (MD15G1221400), MdARF20 (MD13G1234500), MdARF21 (MD16G1239300), MdARF105 (MD07G1152100), MdARF111 (MD14G1131900), and MdActin (XM_029089583.1).
Supplementary Material
Web_Material_uhaf290
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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